Utility of the combination of hederagenin glucoside saponins and chromane hydrazone in the topical treatment of canine cutaneous leishmaniasis. An observational study.

Canine cutaneous leishmaniasis (CCL) is an emerging zoonotic infection endemic in several countries of the world. Due to variable response to therapy and frequency of relapses, a more effective, safer, and inexpensive treatment is needed. Recently, it was reported that the hederagenin glucoside saponins (SS) and chromane-derived hydrazone (TC2) combined in a 1:1 ratio has high potential in antileishmanial therapy since both compounds alter the survival of Leishmania and the ability to infect adjacent macrophage. Not only the skin permeation and the absorption of an ointment containing 2% TC2 and 2% SS (w/w) was determined in this work, but also the acute dermal toxicity in both in vitro and in vivo assays. Last, the effectiveness and safety of the topical therapy with 2% TC2-2% SS ointment was evaluated in an observational study in dogs with diagnosis of cutaneous leishmaniasis (CL). Both TC2 and SS diffused through pig ear skin and traces of TC2 (but not SS) were detected in the stratum corneum of mice at 6-24 h. Neither TC2 nor SS was detected in plasma. The acute dermal toxicity was negative. Treatment with 2% TC2-2% SS ointment produced a complete long-term clinical cure in 56 dogs (24 females and 32 males) from the Orinoco and Amazonas regions in southeastern Colombia without adverse effects. All dogs have remained disease-free for the last 24 months. In conclusion, these results support the use of this topical therapy as a safer and new first-line local treatment of CCL that could help limit the spread of CL from dogs to humans.

Withanolides from Whitania aristata and their diuretic activity.

AIM OF THE STUDY: Withania aristata is an endemic plant used traditionally in Canary Islands as a diuretic. In this paper, we report on this pharmacological activity in several extracts of the dry vegetal material collected and the identification and diuretic activity of two withanolides, one of them previously not reported, isolated from the most active fraction. MATERIAL AND METHODS: Four Whitania aristata extracts at 100 mg/kg were orally administered to laboratory animals to evaluate their diuretic activity. From the most active fraction, two withanolides were isolated. Both and a mixture of them at 5 and 10 mg/kg were analyzed too as diuretics. Water excretion rate and content of Na(+) and K(+) electrolytes were measured in the urine of saline-loaded animals. RESULTS: Whitania aristata water fraction, the two withanolides and the mixture of these compounds displayed high diuretic activity, with a significant excretion of sodium and potassium ions in laboratory animals. CONCLUSIONS: This research supports the ethno-medicinal use of Whitania aristata as diuretic. This activity seems to be associated to the presence of a new type of natural diuretic agents, such as withaferin A and witharistatin.

Delignificación selectiva del pasto pennisetum purpureum x pennisetum typhoides usando basidiomicetos ligninolíticos / Selective delignification of the pennisetum purpureum x pennisetum typhoides grass using ligninolytic basidiomycetes

Muestras de pasto Pennisetum purpureum x Pennisetum typhoides de 120 días de corte se someten durante siete semanas a fermentación selectiva en estado sólido (FES) con cepas de Ganoderma ssp y Lentinus ssp. Se realiza la caracterización de la delignificación por Infrarrojo con Transformada de Fourier (IR-TF) midiendo las áreas de las principales bandas características. Mediante esta técnica se establece que las muestras tratadas con Ganoderma spp se obtiene una pérdida del 70 por ciento de los compuestos aromáticos con relación a los alifáticos. En las semanas cero y séptima se establecieron valores de lignina en detergente ácido (LDA) 55.9 por ciento y 10.7 por ciento, respectivamente. En este mismo periodo los contenidos de materia seca y celulosa variaron del 73.3 por ciento al 92.9 por ciento y del 3.1 por ciento al 51.7 por ciento respectivamente. Estos resultados confirman una degradación selectiva de la lignina en las muestras tratadas con Ganoderma spp y medios suplementados con manganeso. Las pruebas de degradabilidad in situ, utilizando la técnica de la bolsa de nailon y de digestibilidad in vitro de la materia seca basada en técnicas enzimáticas y gravimétricas, no mostraron mejoramiento de la digestibilidad del pasto como consecuencia de la fermentación con las dos cepas de hongos basidiomicetos, corroborando lo indicado por otros autores que afirman que los hongos puede ser tóxicos para la microflora del rumen y, por lo tanto, pueden la digestibilidad de la materia seca

The human olfactory receptor repertoire.

BACKGROUND: The mammalian olfactory apparatus is able to recognize and distinguish thousands of structurally diverse volatile chemicals. This chemosensory function is mediated by a very large family of seven-transmembrane olfactory (odorant) receptors encoded by approximately 1,000 genes, the majority of which are believed to be pseudogenes in humans. RESULTS: The strategy of our sequence database mining for full-length, functional candidate odorant receptor genes was based on the high overall sequence similarity and presence of a number of conserved sequence motifs in all known mammalian odorant receptors as well as the absence of introns in their coding sequences. We report here the identification and physical cloning of 347 putative human full-length odorant receptor genes. Comparative sequence analysis of the predicted gene products allowed us to identify and define a number of consensus sequence motifs and structural features of this vast family of receptors. A new nomenclature for human odorant receptors based on their chromosomal localization and phylogenetic analysis is proposed. We believe that these sequences represent the essentially complete repertoire of functional human odorant receptors. CONCLUSIONS: The identification and cloning of all functional human odorant receptor genes is an important initial step in understanding receptor-ligand specificity and combinatorial encoding of odorant stimuli in human olfaction.

Passifloricins, polyketides alpha-pyrones from Passiflora foetida resin.

Three polyketides alpha-pyrones, named passifloricins, were isolated from Passiflora foetida resin; their structures and relative configurations were assigned through 2D NMR spectroscopic analyses. These types of compounds were not detected in other passion flowers.

A 'non-canonical' DNA-binding element mediates the response of the Fas-ligand promoter to c-Myc.

Cell number is regulated by maintaining a balance between cell proliferation and cell death through apoptosis. Key regulators of this balance include the oncogene product c-Myc, which promotes either entry into the cell cycle or apoptosis [1]. Although the mechanism of c-Myc-induced apoptosis remains unclear, it is susceptible to regulation by survival factors [2,3] and can proceed through the interaction of Fas ligand (FasL) with its receptor, Fas [4]. Activated T lymphocytes are eliminated by an apoptotic process known as activation-induced cell death (AICD), which requires the transcriptional induction of FasL expression [5-7] and sustained levels of c-Myc [8]. The FasL promoter can be driven by c-Myc overexpression, and functional inhibitors of Myc and its binding partner, Max, inhibit the transcriptional activity of the FasL promoter [9,10]. We identified a non-canonical binding site (ATTCTCT) for c-Myc-Max heterodimers in the FasL promoter, which, when mutated, abolished activity in response to c-Myc. Exchange of the canonical c-Myc responsive elements (CACGTG) in the ornithine decarboxylase (ODC) promoter [11] with the non-canonical sequence in the FasL promoter generated an ODC-FasL promoter that was significantly more responsive to c-Myc than the wild-type ODC promoter. Our findings identify a precise physiological role for c-Myc in the induction of apoptosis as a transcriptional regulator of the FasL gene.

Isolation of (S)-(+)-naproxene from Musa acuminata. Inhibitory effect of naproxene and its 7-methoxy isomer on constitutive COX-1 and inducible COX-2.

The isolation and characterisation of (S)-(+)-6-methoxy-alpha-methyl-2-naphthaleneacetic acid, a well known synthetic non-steroidal anti-inflammatory drug (naproxene), from a natural source is described for the first time. We evaluated the ability of naproxene and its 7-methoxy isomer to abrogate constitutive COX-1 and inducible COX-2 activity in human A549 cells. Naproxene inhibited COX-1 (IC50 = 3.42 microM) and COX-2 (IC50 = 1.53 microM), whereas the 7-methoxy isomer had no appreciable effect on COX-1 (IC50 >> 100 microM) but also abrogated the activity of COX-2 enzyme (IC50 = 14.42 microM).

Expression of Fas ligand in activated T cells is regulated by c-Myc.

The transcription factor c-Myc is important for the control of cell cycle progression, neoplasia, and apoptotic cell death. c-Myc dimerizes with its partner Max to form an active transcription factor complex. Little is known, however, about the transcriptional targets of c-Myc and their roles in c-Myc-induced cell death. Here we demonstrate that T cell activation-induced expression of Fas ligand (FasL, CD95-L, APO-1-L), which can induce apoptotic cell death in many different cell types, is regulated by c-Myc. Down-modulation of c-Myc protein via antisense oligonucleotides blocked activation-induced FasL mRNA and protein expression and functional FasL expression in activated T cells and T cell lines. Further, FasL promoter activity in T cells is driven by overexpression of c-Myc and inhibited by expression of dominant-negative mutants of c-Myc and Max. Our findings indicate that c-Myc controls apoptotic cell death in T cells through regulation of FasL expression.

Caspase-3 is the primary activator of apoptotic DNA fragmentation via DNA fragmentation factor-45/inhibitor of caspase-activated DNase inactivation.

Caspase-3 initiates apoptotic DNA fragmentation by proteolytically inactivating DFF45 (DNA fragmentation factor-45)/ICAD (inhibitor of caspase-activated DNase), which releases active DFF40/CAD (caspase-activated DNase), the inhibitor's associated endonuclease. Here, we examined whether other apoptotic proteinases initiated DNA fragmentation via DFF45/ICAD inactivation. In a cell-free assay, caspases-3, -6, -7, -8, and granzyme B initiated benzoyloxycarbonyl-Asp-Glu-Val-Asp (DEVD) cleaving caspase activity, DFF45/ICAD inactivation, and DNA fragmentation, but calpain and cathepsin D failed to initiate these events. Strikingly, only the DEVD cleaving caspases, caspase-3 and caspase-7, inactivated DFF45/ICAD and promoted DNA fragmentation in an in vitro DFF40/CAD assay, suggesting that granzyme B, caspase-6, and caspase-8 promote DFF45/ICAD inactivation and DNA fragmentation indirectly by activating caspase-3 and/or caspase-7. In vitro, however, caspase-3 inactivated DFF45/ICAD and promoted DNA fragmentation more effectively than caspase-7 and endogenous levels of caspase-7 failed to inactivate DFF45/ICAD in caspase-3 null MCF7 cells and extracts. Together, these data suggest that caspase-3 is the primary inactivator of DFF45/ICAD and therefore the primary activator of apoptotic DNA fragmentation.

DNA damaging agents induce expression of Fas ligand and subsequent apoptosis in T lymphocytes via the activation of NF-kappa B and AP-1.

Apoptosis induced by DNA damage and other stresses can proceed via expression of Fas ligand (FasL) and ligation of its receptor, Fas (CD95). We report that activation of the two transcription factors NF-kappa B and AP-1 is crucially involved in FasL expression induced by etoposide, teniposide, and UV irradiation. A nondegradable mutant of I kappa B blocked both FasL expression and apoptosis induced by DNA damage but not Fas ligation. These stimuli also induced the stress-activated kinase pathway (SAPK/JNK), which was required for the maximal induction of apoptosis. A 1.2 kb FasL promoter responded to DNA damage, as well as coexpression with p65 Rel or Fos/Jun. Mutations in the relevant NF-kappa B and AP-1 binding sites eliminated these responses. Thus, activation of NF-kappa B and AP-1 contributes to stress-induced apoptosis via the expression of FasL.

Somatic mutations in the p53 tumor suppressor gene in rheumatoid arthritis synovium.

The factors that regulate the perpetuation and invasiveness of rheumatoid synovitis have been the subject of considerable inquiry, and the possibility that nonimmunologic defects can contribute to the disease has not been rigorously addressed. Using a mismatch detection system, we report that synovial tissue from the joints of severe chronic rheumatoid arthritis patients contain mutant p53 transcripts, which were not found in skin samples from the same patients or in joints of patients with osteoarthritis. Mutant p53 transcripts also were identified in synoviocytes cultured from rheumatoid joints. The predicted amino acid substitutions in p53 were identical or similar to those commonly observed in a variety of tumors and might influence growth and survival of rheumatoid synoviocytes. Thus, mutations in p53 and subsequent selection of the mutant cells may occur in the joints of patients as a consequence of inflammation and contribute to the pathogenesis of the disease.

Danielone, a phytoalexin from papaya fruit.

A new phytoalexin was induced and isolated from papaya fruit slices treated with copper salts; its structure was established as 3',5'-dimethoxy-4'-hydroxy-(2-hydroxy)acetophenone. This compound showed high antifungal activity against Colletotrichum gloesporioides, a pathogenic fungus of papaya.

Immunoregulatory activity of a T-cell receptor alpha chain demonstrated by in vitro transcription and translation.

Previous studies from our laboratory and those of others suggested the possibility that the T-cell antigen receptor alpha (TCR alpha) chain from some T cells can be released in a soluble form and can have antigen-specific immunoregulatory activity. We have analyzed this phenomenon by in vitro transcription and translation (IVTT) of a cDNA encoding a TCR alpha chain (A1.1 TCR alpha) suspected of having such activity. We found that TCR alpha, but not TCR beta, protein produced in this way showed antigen-specific regulatory activity in an in vitro immune-response assay. Protein derived from truncated forms of the A1.1 TCR alpha cDNA had activity providing that, in addition to the variable (V) and joining (J) regions of the alpha chain (VJ alpha), at least the first 25 amino acids of the alpha chain of the constant (C) region (C alpha) were present. Addition of an irrelevant protein sequence to the VJ alpha failed to impart activity to the molecule, suggesting that the C alpha requirement is not simply for stabilization of the resulting protein. These results are discussed in the context of other recent studies on the immunoregulatory activity of soluble TCR alpha molecules, and the possible physiological relevance of these observations is considered.

Cell-autonomous Fas (CD95)/Fas-ligand interaction mediates activation-induced apoptosis in T-cell hybridomas.

A number of murine T-cell hybridomas undergo apoptosis within a few hours of activation by specific antigens, mitogens, antibodies against the T-cell antigen receptor, or a combination of phorbol ester and calcium ionophore. This phenomenon has been extensively studied as a model for clonal deletion in the immune system, in which potentially autoreactive T cells eliminate themselves by apoptosis after activation, either in the thymus or in the periphery. Here we show that the Fas/CD95 receptor, which can transduce a potent apoptotic signal when ligand, is rapidly expressed following activation of T-cell hybridomas, as is its functional, membrane-bound ligand. Interference with the ensuing Fas/Fas-ligand interaction inhibits activation-induced apoptosis. Because T-cell receptor ligation can induce apoptosis in a single T hybridoma cell, we suggest that the Fas/Fas-ligand interaction can induce cell death in a cell-autonomous manner.

Promotion and inhibition of activation-induced apoptosis in T-cell hybridomas by oncogenes and related signals.

The Two Signal: Death/Survival Model suggests that cellular proliferation and physiological cell death should be intimately associated such that, in the absence of external influences, a normal cell departing from rest will have an equal probability of undergoing either process. The c-Myc protooncogene product has been implicated in cell cycle progression and in the control of gene expression, and more recently c-Myc has also been seen to promote apoptotic cell death. As predicted from the model, c-Myc-induced apoptosis is inhibited by growth factors or other anti-apoptotic signals including those provided by some oncogenes. Here, we discuss experiments that test the Two Signal: Death/Survival Model in the phenomenon of activation-induced apoptosis in T-cell hybridomas. Ligation of the antigen receptor on these cells leads to activation, resulting in cytokine production and apoptosis. Inhibition of c-Myc expression by addition of antisense oligodeoxynucleotides or transforming growth factor beta inhibits this form of apoptosis. Because c-Myc is known to bind to several cellular proteins, including Max, we further examined the effects of expression of a dominant negative Max on activation-induced apoptosis. We found that this Max mutant, which interferes with the function of the Myc/Max heterodimer, inhibits the induction of apoptosis by antigen receptor ligation. Thus, both Myc and Max play roles in activation-induced apoptosis, presumably via control of gene expression. Further, as predicted, signals generated from growth factor receptors or the v-Abl oncogene interfere with activation-induced apoptosis. In contrast, the anti-apoptotic effects of Bcl-2 are not active in this form of apoptosis. Finally, a role for Fas/Fas-ligand interactions in activation-induced apoptosis is considered.

The structure of acnistin B and the immunosuppressive effects of acnistins A, B, and E.

The structure of the new withanolide:acnistin B, isolated from Dunalia solanacea, has been established by NMR experiments and the immunosuppressive effects of acnistins A, B and E on human lymphocytes have been measured.

Unambiguous 13C NMR assignment of acnistins and absolute configuration of acnistin A.

Carbon and proton atoms were fully assigned in this new type of withanolide by HMQC and HMBC experiments. The absolute configuration of acnistin A was determined by X-ray diffraction. Proton and 13C NMR measurements are particularly useful in identifying members of this group of natural products.

Apoptotic cell death induced by c-myc is inhibited by bcl-2.

Apoptosis is a form of physiological cell death, characterized by chromatin condensation, cytoplasmic blebbing and DNA fragmentation, which often depends on RNA and protein synthesis by the dying cell. The c-myc proto-oncogene, usually implicated in cell transformation, differentiation and cell-cycle progression also has a central role in some forms of apoptosis. These opposing roles of myc in cell growth and death require that other gene products dictate the outcome of c-Myc expression on a cell. A candidate for such a modifying gene is bcl-2, whose product prolongs cell survival and blocks apoptosis in some systems. Here we demonstrate that Bcl-2 prevents apoptotic death induced by c-Myc, provide a mechanism whereby cells can express c-Myc without undergoing apoptosis, and give a possible explanation for the ability of Bcl-2 to synergize with c-Myc in cell transformation.

The induction of apoptosis by chemotherapeutic agents occurs in all phases of the cell cycle.

A1.1 T-cell hybridoma cells exposed to either actinomycin D (1 microgram/ml), camptothecin (200 ng/ml) or aphidicolin (10 micrograms/ml) for 16 hrs at 37 degrees C die via apoptosis. The cell death was independent of RNA synthesis, in contrast to previous data reported for other forms of apoptosis in murine lymphocyte cells and their derived lines. Each of the three agents described appeared to induce death in all phases of the cell cycle in asynchronously proliferating cells. G1 cells appeared to be more susceptible to the effects of camptothecin and contrasts with other reports which detail its selectivity for S and G2 phase cells. This might indicate that cells are progressing into S phase before dying or, alternatively, cells may indeed be dying in G1. When elutriated synchronised cells were exposed to each of the three cytotoxic agents cell death occurred in all phases of the cell cycle. In view of the fact that G1 and S phase cells did not cycle to any appreciable extent during drug exposure, it was likely that ensuing death, occurred specifically from these phases. G2/M cells, however, moved rapidly into G1 in the presence of each drug, thus making it difficult to determine whether G2/M cells were capable of undergoing drug-induced apoptosis. To overcome this problem, nocodazole (50 ng/ml) was used to block asynchronous cells in M phase. When these cells were exposed to actinomycin D, aphidicolin or camptothecin, cell death ensued via apoptosis.